Materials and methods used in the study 1 Specimens used

Specimens were collected from the Pathology archives from two groups of patients treated at the Middlesex and University College Hospitals, London, between 1979 and 1981. The patients were matched for similar clinical features, pathology and treatment but had either long or short-term survival after presentation. In the short-term survival group, the primary cancers progressed to form distant métastasés within five years and all the patients died within seven years of primary surgery. In the long-term survival group, there was no sign of cancer recurrence despite fifteen years follow-up. The clinical and histopathological features of the specimens used are shown summarised in tables 3.1 and 3.2, the individual patient details are shown in appendix 1.

Table 3.1 H istopathological features o f the breast cancer specim ens used

in the pilot study.

Description Short-term survivors Long-term survivors

Histopathological type:

Infiltrating ductal cancer 9 8

Infiltrating ductal and lobular cancer - I

Infiltrating lobular cancer 1 -

Grade:

I I 1

II 1 3

III 5 3

Axillary lymph node involvement:

Lymph node negative 2 5

Lymph node positive 8 4

3.2 2 Patient follow-up to monitor disease progression.

Disease progression was monitored by patient follow-up, this was achieved by examining the patient's medical records and recording relevant information. Patients typically attended the hospital every three months for the first year post- operatively, every six months for the following year and then yearly until ten years had elapsed. Patients with métastasés may have attended at other times if the disease was symptomatic. Metastatic spread was recorded only if it had been identified clinically and by further investigation, for example. X-ray in the case of métastasés to lung or bone. Cases where a patient had died from metastatic

disease were confirmed by death certificate from the Office o f Population Census and Surveys. In the case of patients who had been discharged with more than ten years disease free survival, their General Practitioner was contacted to confirm that the patient was still alive with no clinical symptom or sign of local or distant recurrence.

Table 3.2 Clinical features o f the breast cancer specim ens used.

Description Short-term survivors Long-term survivors

age at presentation in years (mean) 64 58

range = 5 3 -7 6 53 -6 8

size o f tumour in cm (mean) 2.9 2

range = 1 - 6 1 - 6

wide local excision - 1

Mastectomy 10 8

follow-up information:

no sign of recurrence in 15 years - 9

widespread métastasés within 5 years 10 -

3.2.3 Macrodissection o f the specimens.

Breast tumour tissue contains a heterogeneous mixture of cancer, normal cells and reactive tissue in different amounts. We removed as much o f the non-tumour areas as possible in order to limit sample-to-sample variation. In each case, a 5pm thick section was cut from the paraffin-wax embedded breast cancer specimen and mounted onto a glass slide. The section was dewaxed by passing through xylene and graded alcohols to water then stained with haematoxylin and eosin to identify the different cell types. The specimens were dissected on the paraffin-wax blocks by comparing the haematoxylin and eosin stained section with the paraffin-wax block and carefully removing areas of non-malignant cells, necrosis or lymphocyte infiltration. A number of specimens were stained with haematoxylin and eosin after macrodissection to confirm that the areas of non- malignant cells had been effectively removed by the procedure. A size 22 scalpel was used for the macrodissection step.

3.2.4 Preparation o f cancer specimens fo r oligosaccharide release.

Glycoproteins were prepared from a pool of twenty 5 pm thick sections cut from each of the macro-dissected paraffin-wax embedded tissues, using the methods detailed in section 2.2.4.

3.2.5 Oligosaccharide release and labelling.

Oligosaccharides were released using the methods detailed in section 2.2.5, but because only two specimens could be processed at the same time in the GlycoPrep 1000, the oligosaccharide release procedure was performed manually on batches of six specimens. 2 mg of freeze-dried fetuin from fetal calf serum (Sigma) was included to check that oligosaccharide release was proceeding in a predictable manner. The freeze-dried weight of breast cancer glycoproteins was not determined accurately since the single milligram quantities had previously been determined on other breast cancer specimens, to check solubilisation in hydrazine. In addition, there were difficulties associated with weighing such small quantities of lyophilised proteins since freeze-dried material takes up atmospheric moisture and this makes such measurements both difficult and inaccurate. By reference to previous preparations from breast cancer specimens of a similar size, it was predicted that a maximum o f 2 mg of glycoprotein had been prepared from each of the specimens. To release the oligosaccharides 800 pi o f anhydrous hydrazine (Oxford Glycosciences) was added. The volume of hydrazine used was selected to ensure that there was an excess of hydrazine present. The hydrazinolysis reaction was allowed to proceed under a blanket of argon at 95°C for 18 hours. Other steps from the method of Takasaki et al., (1982) were followed. Residual peptides were removed as detailed in section 2.2.6 and the oligosaccharides were labelled with 2-AB as detailed in section 2.2.7.

3.2.6 Analysis o f the oligosaccharides.

The oligosaccharide pool from each of the breast cancer specimens was reconstituted in 20 pi of HPLC grade water.

3.2.6.1 Sialylated oligosaccharides.

A 10 pi aliquot of the oligosaccharide pool was separated by anion- exchange chromatography on a GlycoSep C column as detailed in section 2.2.8.

3.2.6. 2...Neutral oligosaccharides.

The remaining 10 |li1 aliquot of the oligosaccharide pool was mixed with 20 |il of dextran ladder and separated by gel permeation chromatography (GPC) on a BioGel P4 column, using the instrument and conditions described in section 2.2.8.

3.2.6. 3...Separation o f monosaccharides using a Carbo Pak PAIOO column.

The GPC fractions corresponding to less than 3 glucose units hydrodynamic volume were collected from one of the long-term and one of the short-term survival patients. The fractions were dried by centrifugal evaporation and the monosaccharide structures analysed by separation on a CarboPak PAIOO column (Dionex). The analysis was performed by Miss Heidi Lacey as follows: column flow rate was set to 0.5 ml / min and the column was washed with 20 column volumes of 1 mol /1 sodium hydroxide. The column was equilibrated in 160 mmol /1 sodium hydroxide and the unknown

monosaccharide loaded onto the column in the same buffer. The elution position of the unknown monosaccharides from the GPC separations were compared with the elution positions of 2-AB labelled GalNAc, GlcNAc, Glc and Gal.

3,2.7 Analysis o f the results.

The peaks on the chromatograms corresponding to fluorescently labelled oligosaccharides were integrated as detailed in section 2.2.8.1.3. The proportion o f neutral to sialylated oligosaccharides, and the diversity of sialylated oligosaccharides were compared using the Student /-test (non­ paired test). ‘Significant’ differences were recorded for values of P which were less than 0.05, ‘suggestively significant’ values were recorded for values of P which were less than 0.1 but greater than 0.05. Values of P which were greater than 0.1 were recorded as ‘not significantly different’.